Project: mtDNA Deletions and Focal Respiratory Chain Deficiency - Mitochondria are the major source of energy in cells. They contain their own DNA (mtDNA) whose genes encode components of the respiratory chain. They are maternally inherited and are absolutely critical for the function of those tissues that are highly dependent on aerobic metabolism, such as muscle and brain. In the last eight years, mtDNA mutations have been discovered in a number of neuromuscular disorders, including progressive external ophthalmoplegia (PEO), Kearns-Sayre syndrome (KSS), myoclonus epilepsy with ragged-red fibers (MERRF), and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). We propose to gain further insight into the pathogenesis of respiratory chain enzyme deficiency in individual muscle fibers and neurons of KSS and PEO patients with mtDNA deletions, using three different approaches. First, we will determine the cellular distribution of multiple mtDNA deletions in muscle from patients with mendelian-inherited PEO, and correlate these observations with different cellular phenotypes. To study this question, we will carry out in situ hybridization and single-fiber PCR studies in parallel with histochemical and immunohistochemical analysis of the samples. This approach will allow us to draw genotyp-phenotype correlations at the individual fiber level. Second, we will study subunits of the respiratory chain in brain from patients with sporadic KSS, and for comparative purposes MERRF and MELAS, in order to determine the potential impairment in the expression of mtDNA-encoded polypeptides in specific brain regions that may account for neuronal dysfunction. To investigate this question, we will carry out immunohistochemical studies on paraffin-embedded brain sections in conjunction with conventional neuropathological evaluation of the samples. Third, we will study the neuroanatomical distribution of mtDNA deletion in brain from patients with sporadic KSS in order to determine if there are accumulations of mutated mtDNAs in specific brain regions, using in situ hybridization in situ PCR methodologies. Because the clinical manifestations of mitochondrial encephalomyopathies are dominated by signs and symptoms of brain involvement, including ataxia, seizures, and dementia, molecular and immunohistochemical studies on affected brains may provide further understanding of neuronal dysfunction in specialized brain regions. This may clarify pathogenetic mechanisms and help us devise rational therapeutic approaches.